The green fluorescent protein (GFP: Green Fluorescent Protein) derived from a jellyfish, Aequorea Victoria, or a modified protein thereof is capable of recombinant expression in heterogeneous cells especially in various kinds of mammalian cells, and the obtained recombinant protein exhibits fluorescence performance in host cells. Using this feature, it has been attempted to use GFP derived from A. victoria and homologues thereof for various objects and applications as an in vivo fluorescent marker protein in the field of biochemistry, cell physiology and medicine (See Reference 1: Lippincott-Schwartz, J. G. H. Patterson, Science Vol. 300, 87-91 (2003); Reference 2: Tsien, R. Y., Annu. Rev. Biochem. Vol. 67, 509-544 (1998)).
In addition, besides GFP derived from A. victoria, GFP-like proteins have been cloned from class Hydrozoa of phylum Cnidaria (Cnidaria) and further GFP-like proteins have been also cloned from class Anthozoa of phylum Cnidaria.
Concerning these GFP-like proteins discovered in class Anthozoa of phylum Cnidaria, it is reported that they probably constitute a fluorescent protein family having bioevolutionarily the common origin (see Reference 3: Y. A. Labas et al., Proc. Natl. Acad. Sci. U.S.A. Vol. 99, 4256-4261 (2002)).
Concerning GFP derived from A. victoria, researches on the mechanism being essential to the exhibition of the fluorescence performance therein have progressed. First, it was revealed that in the process for folding into the natural steric structure, through which translated GFP polypeptide was converted into mature GFP having the fluorescence performance through the steps of cyclization of internal tripeptide site and subsequent oxidization thereto, which resulted in formation of a fluorophore. Furthermore, it has been also confirmed that SYG, 65-67th residues in the deduced amino acid sequence of wild type GFP derived from A. victoria is the internal tripeptide site, which forms a fluorophore. For example, it has been reported that a blue shift as compared with green fluorescence of wild type GFP is caused in the fluorescence in Y66H-GFP, in which mutation of Tyr to His at the 66th residue was made, showing blue fluorescence with a maximum at the wavelength of 448 nm. Furthermore, in S65T-GFP, where mutation of Ser to Thr at the 65th residue was made, the wavelength of a maximum of the fluorescence thereof was 510 nm, showing a slight red shift as compared with green fluorescence of wild type GFP. It has been also reported that formation of fluorophore, which was achieved through cyclization of an internal tripeptide: TYG site and subsequent oxidization, proceeds significantly more quickly in S65T-GFP than in SYG of wild type GFP.
Besides the aforementioned introduction of a mutation into the 65-67th SYG site, it has been also reported that when mutations of T203H, T203F and T203Y, which respectively replaces Thr with His, Phe, and Tyr at the 203rd position in the wild type GFP derived from A. Victoria, are introduced, the wavelength for the maximum in the fluorescence thereof shows a remarkable red shift to about 530 nm, resulting in yellow fluorescent protein (YFP: Yellow Fluorescent Protein). Moreover, it has been reported that EGFP (“enhanced” GFP), in which mutation of F64L replacing Phe with Leu at the 64th position adjacent to the 65-67th SYG site is made, exhibits a markedly improved maturation process accompanied by formation of fluorophore as compared with wild type GFP (see Reference 4: B. P. Cormack et al., Gene Vol. 173, 33-38 (1996)).
In this way, with regard to GFP-like proteins derived from various sea animals belonging to the phylum Cnidaria represented by GFP derived from A. victoria, a number of attempts utilizing them as an in vivo fluorescent marker protein, which can be expressed in an animal cell, have been made. In the meantime, it is known that there exist lots of marine organisms, especially animal planktons which show bioluminescence. Accordingly, the existence of novel fluorescent proteins is demanded which constitute another type of protein family having a bio-evolutionarily different origin from the fluorescent protein family to which GFP derived from A. victoria belongs. Thus, search for a new fluorescent protein family is desired, which can be used as an in vivo fluorescent marker protein which can be expressed in a host animal cell.